夹心ELISA定量检测猪圆环病毒2b型抗原方法的建立
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摘要
为建立快速定量检测猪圆环病毒2b型(PCV2b)抗原含量的方法,本研究采用重组抗PCV2b纳米抗体作为捕获抗体,鼠抗PCV2b单克隆抗体为检测抗体,建立了定量检测PCV2b的夹心ELISA方法。该方法不仅能够用于猪圆环疫苗中PCV2b抗原的检测,还可用于大肠杆菌或杆状病毒表达的PCV2b衣壳蛋白(Cap)亚单位疫苗的检测,且与其它猪病毒无交叉反应。该方法检测细胞培养PCV2b的线性范围是104.3TCID50/mL~105.5TCID50/mL,样品回收率为89.3%~105.6%,该方法批内、批间变异系数小于5%,重复性好。该方法与间接免疫荧光方法(IFA)检测相同样品时误差值小于10%,并且ELISA检测结果离散度更小。分别用两种方法定量PCV2b抗原,取相同抗原量免疫猪,其诱导产生的PCV2b抗体效价无显著差异。本研究构建的夹心ELISA方法在定量猪圆环疫苗中PCV2b抗原方面,与传统的IFA方法比较其显示了良好的符合性,并具有更好的重复性和更短的检测周期,是替代IFA方法用于PCV2b定量检测的更优选择。
To develop an assay for quantitative determination porcine circovirus type 2b(PCV2b), a sandwich ELISA was established using a his-tagged nanobody as capture antibody and a mouse monoclonal antibody against PCV2b as detection antibody. This assay was specific for detection of PK-15 cell proliferated PCV2b and E.coli or Baculovirus expressed PCV2b Cap protein but had no cross-reaction with other swine virus. The linear range of this assay was determined to be 104.3 TCID50/mL to105.5 TCID50/mL for PCV2b and the recovery ratio of spiked PCV2 b was 89.3%-105.6%. The coefficient variation(CV) of intraand inter-assay were both less than 5%, demonstrating the high repeatability of the assay. Compared with indirect immunofluorescent assay(IFA), the virus titers quantified by this ELISA and IFA had no significant difference, and the dispersion was smaller than the latter. The serological response of pigs immunized with PCV2b inactivated vaccine also indicated that the antigen quantified by ELISA was reliable. This assay showed high reproducibility and a short detection time, and it could be used to determine the antigen yields for PCV2b vaccines production.
To develop an assay for quantitative determination porcine circovirus type 2b(PCV2b), a sandwich ELISA was established using a his-tagged nanobody as capture antibody and a mouse monoclonal antibody against PCV2b as detection antibody. This assay was specific for detection of PK-15 cell proliferated PCV2b and E.coli or Baculovirus expressed PCV2b Cap protein but had no cross-reaction with other swine virus. The linear range of this assay was determined to be 104.3 TCID50/mL to105.5 TCID50/mL for PCV2b and the recovery ratio of spiked PCV2 b was 89.3%-105.6%. The coefficient variation(CV) of intraand inter-assay were both less than 5%, demonstrating the high repeatability of the assay. Compared with indirect immunofluorescent assay(IFA), the virus titers quantified by this ELISA and IFA had no significant difference, and the dispersion was smaller than the latter. The serological response of pigs immunized with PCV2b inactivated vaccine also indicated that the antigen quantified by ELISA was reliable. This assay showed high reproducibility and a short detection time, and it could be used to determine the antigen yields for PCV2b vaccines production.
引文
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[12]刘湘涛,杨顺利,尹双辉,等.抗猪圆环病毒2型的双峰驼重链单域抗体及其制备方法和用途[P].中国201210249621.4,2012-07-18.
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[14]赵燕.猪圆环病毒2型疫苗的研究进展[J].畜禽业,2016,(01):30-34.
[15]M·艾希梅厄,G·尼策尔,M·谢弗.PCV2免疫原性组合物和产生这种组合物的方法[P].中国200580047741.4,2005-12-29.
[2]Chae C.A review of porcine circovirus 2-associated syndromes and diseases[J].Vet J,2005,169(3):326-336.
[3]Blanchard P,Mahe D,Cariole T R,et al.Protection of swine against post-weaning multisystemic wasting syndrome(PMWS)by porcine circovirus type 2(PCV2)proteins[J].Vaccine,2003,21(31):4565-4575.
[4]Rooijakkers E,Groen J,Uittenbogarrd J,et al.Development and evaluation of alternative testing methods for the in vivo NIHpotency test used for the quality control of inactivated rabies vaccines[J].Dev Biol Stand,1996,86:137-145.
[5]Maas R A,de Winter M P M,Venema S,et al.Antigen quantification as in vitro alternative for potency testing of inactivated viral poultry vaccines[J].Vet Q,2000,22(4):223-227.
[6]Pecora A,Aguirreburualde M S P,Rodriguez D,et al.Development and validation of an ELISA for quantitation of bovine viral diarrhea virus antigen in the critical stages of vaccine production[J].J Virol Methods,2009,162(1-2):170-178.
[7]Saravanan P,Sen A,Balamurugan V,et al.Rapid quality control of a live attenuated Peste des petits ruminants(PPR)vaccine by monoclonal antibody based sandwich ELISA[J].Biologicals,2008,36(1):1-6.
[8]黄立平,危艳武,刘长明.猪圆环病毒2型抗原捕获ELISA试剂盒[P].中国201510030198.2,2015-01-21.
[9]翟淑燕,陈兴慧,白娟,等.猪圆环病毒2型Cap蛋白单克隆抗体的制备与应用[J].中国兽医科学,2014,(10):1022-1028.
[10]李文良,王先炜,马涛,等.表达猪圆环病毒2型SH1分离株Cap蛋白的重组杆状病毒的构建与鉴定[J].中国兽医科学,2010,(11):1156-1160.
[11]赵晓云,乔绪稳,陈瑾,等.利用E.coli表达猪圆环病毒2型Cap蛋白生产病毒样颗粒疫苗[J].中国农业科学,2015,(05):976-986.
[12]刘湘涛,杨顺利,尹双辉,等.抗猪圆环病毒2型的双峰驼重链单域抗体及其制备方法和用途[P].中国201210249621.4,2012-07-18.
[13]Nawagitgul P,Morozov I,Bolin S R,et al.Open reading frame2 of porcine circovirus type 2 encodes a major capsid protein[J].J Gen Virol,2000,81(Pt 9):2281-2287.
[14]赵燕.猪圆环病毒2型疫苗的研究进展[J].畜禽业,2016,(01):30-34.
[15]M·艾希梅厄,G·尼策尔,M·谢弗.PCV2免疫原性组合物和产生这种组合物的方法[P].中国200580047741.4,2005-12-29.